Method and composition for reconforming multi-epitopic antigens to initiate an immune response

ABSTRACT

The invention concerns methods and compositions for intiating and/or enhancing an immune response by contacting a binding reagent with a soluble antigen, wherein the binding reagent-antigen pair generates an immune response to the antigen.

TECHNICAL FIELD

[0001] The invention concerns methods and compositions for initiatingand/or enhancing an immune response in vivo.

BACKGROUND ART

[0002] All vertebrates have an immune system. The ability of vertebratesto protect themselves against infectious microbes, toxins, viruses, orother foreign macromolecules is referred to as immunity. Immunity ishighly specific; such specificity is a fundamental characteristic ofimmune responses. Many of the responses of the immune system initiatethe destruction and elimination of invading organisms and any toxicmolecules produced by them. Because the nature of these immune reactionsis inherently destructive, it is essential that the response isprecisely limited to the foreign molecules and not to those of the hostitself. This ability to distinguish between foreign molecules and selfmolecules is another fundamental feature of the immune system.

[0003] The art distinguishes between natural and acquired or specificimmunity. Natural immunity is comprised of defense mechanisms which areactive before exposure to microbes or foreign macromolecules, are notenhanced by such exposure, and do not distinguish among most substancesforeign to the body. Effectors of natural immunity are physical barrierssuch as skin or mucous membranes, phagocytic cells such as macrophagesor neutrophils, a class of lymphocytes termed natural killer cells, andthe complement system. Complement is a serum protein complex that isdestructive to certain bacterial and other cells sensitized by specific,complement-fixing antibodies; its activity is effected by a series ofinteractions resulting in proteolytic cleavages and which can follow oneor the other of at least two pathways.

[0004] In vertebrates, the mechanisms of natural and specific immunitycooperate within a system of host defenses, the immune system, toeliminate foreign invaders. In addition to microbes, cancer cells,parasites and virus-infected cells, the immune system also recognizesand eliminates cells or tissues transplanted into a subject from agenetically different individual of the same species (allografts) orfrom a different species (xenografts).

[0005] Acquired or specific immunity comprises defense mechanisms whichare induced or stimulated by exposure to foreign substances. The eventsby which the mechanisms of specific immunity become engaged in thedefense against foreign substances are termed immune responses.Vertebrates have two broad classes of immune responses: antibodyresponses, or humoral immunity, and cell-mediated immune responses, orcellular immunity. Humoral immunity is provided by B lymphocytes, which,after proliferation and differentiation, produce antibodies (proteinsalso known as immunoglobulins) that circulate in the blood-and lymphaticfluid. These antibodies specifically bind to the antigen that inducedthem. Binding by antibody inactivates the foreign substance, e.g., avirus, by blocking the substance's ability to bind to receptors on atarget cell. The humoral response primarily defends against theextracellular phases of bacterial and viral infections. In humoralimmunity, serum alone can transfer the response, and the effectors ofthe response are soluble protein molecules called antibodies.

[0006] The second class of immune responses, cellular immunity, involvethe production of specialized cells, e.g., T lymphocytes, that reactwith foreign antigens on the surface of other host cells. The cellularimmune response is particularly effective against fungi, parasites,intracellular viral infections, cancer cells and other foreign matter.In fact, the majority of T lymphocytes play a regulatory role inimmunity, acting either to enhance or suppress the responses of otherwhite blood cells. These cells, called helper T cells and suppressor Tcells, respectively, are collectively referred to as regulatory cells.Other T lymphocytes, called cytotoxic T cells, kill virus-infectedcells. Both cytotoxic T cells and B lymphocytes are involved directly indefense against infection and are collectively referred to as effectorcells.

[0007] The time course of an immune response is subdivided into thecognitive or recognition phase, during which specific lymphocytesrecognize the foreign antigen; the activation phase, during whichspecific lymphocytes respond to the foreign antigen; and the effectorphase, during which antigen-activated lymphocytes mediate the processesrequired to eliminate the antigen. Lymphocytes are immune cells that arespecialized in mediating and directing specific immune responses. Tcells and B cells become morphologically distinguishable only after theyhave been stimulated by an antigen.

[0008] The immune system has evolved so that it is able to recognizesurface features of macromolecules that are not normal constituents ofthe host. As noted above, a foreign molecule which is recognized by theimmune system (i.e., bound by antibodies), regardless of whether it canitself elicit a response is called an “antigen”, and the portion of theantigen to which an antibody binds is called the “antigenicdeterminant”, or “epitope”. Some antigens, e.g., tumor-associatedantigens such as ovarian cancer or breast cancer antigens, have multipleantibody binding sites. These antigens are termed “multi-epitopic”antigens. When the antigen is a polypeptide, it is customary to classifyepitopes as being linear (i.e., composed of a contiguous sequence ofamino acids repeated along the polypeptide chain) or nonlinear (i.e.,composed of amino acids brought into proximity as a result of thefolding of the polypeptide chain). Nonlinear epitopes are also called“conformational” because they arise through the folding of thepolypeptide chain into a particular conformation, i.e., a distinctive3-D shape. Because of the highly specific nature of the antibody-antigenbond, a primary means of distinguishing between antigens, or betweendifferent epitopes on the same antigen, is by antibody bindingproperties.

[0009] To cope with the immense variety of epitopes encountered, theimmune system of a mammalian individual contains an extremely largerepertoire of lymphocytes, approximately 2×10¹². Each lymphocyte cloneof the repertoire contains surface receptors specific for one epitope.It is estimated that the mammalian immune system can distinguish atleast 10⁸ distinct antigenic determinants. Even a single antigenicdeterminant will, in general, activate many clones, each of whichproduces an antigen-binding site with its own characteristic affinityfor the determinant. Antigens that stimulate the production of hundredsof species of antibodies, each made by a different B cell clone, aresaid to produce a polyclonal response. When only a few clones respond,the response is said to be oligoclonal; when the total response is madeby a single B or T cell clone, the response is said to be monoclonal.The response to most antigens are polyclonal.

[0010] An initial or primary immune response to a foreign antigenenhances the ability of the immune system to respond again to thatantigen. This feature of specific immunity is called immunologic memory,or a secondary immune response. Secondary immune responses are oftenmore effective than primary responses.

[0011] The conventional definition of an antigen is a substance that canelicit in a vertebrate host the formation of a specific antibody or thegeneration of a specific population of lymphocytes reactive with thesubstance. As frequently occurs in science, however, it is now knownthat this definition, although accurate, is not complete. For example,it is now known that some disease conditions suppress or inactivate thehost immune response. Under these conditions, a tumor antigen does notelicit an antibody or generate specific lymphocytes. Thus, not allantigens are capable of eliciting a human immune response.

[0012] The failure in the definition centers on a two-part aspect of theimmune response: the first step in the immune response is therecognition of the presence of a foreign entity; the second step is acomplex array or cascade of reactions, i.e., the response. In the tumorantigen example given above, the immune system can recognize thepresence of a foreign antigen, but it cannot respond. In anotherexample, a failure in the immune system's ability to distinguish betweenself and non-self appears to be at the origin of many autoimmunediseases. Again, this is a failure in recognition, not response.

[0013] As used herein, therefore, if an antigen can be recognized by theimmune system, it is said to be antigenic. If the immune system can alsomount an active response against the antigen, it is said to beimmunogenic. Antigens which are immunogenic are usually macromolecules(such as proteins, nucleic acids, carbohydrates and lipids) of at least5000 Daltons molecular weight. Smaller nonimmunogenic molecules, e.g.,haptens and small antigenic molecules, can stimulate an immune responseif associated with a carrier molecule of sufficient size.

[0014] Antibodies, the effectors of humoral immunity, are secreted byplasma cells, and are among the most abundant components of the blood.Plasma cells are mature end stage cells that appear to have a relativelyshort life span. They are produced when an antigen enters the humanimmune system and, in a complex series of cell interactions, activates Blymphocytes. B lymphocytes then proliferate and differentiate to formplasma cells. Each B lymphocyte is programmed by its DNA to make anantibody molecule of a single specificity. B lymphocytes make twospecial forms of this molecule, one that remains anchored to the outersurface of the cell membrane as a membrane receptor, typically forbinding antigen to the B cell, and one that is secreted.

[0015] Antibodies, also known as immunoglobulins, are proteins. Theyhave two principal functions. The first is to recognize (bind) foreignantigens. The second is to mobilize other elements of the immune systemto destroy the foreign entity.

[0016] The antigen recognition structures of an antibody are variabledomains, and are responsible for antigen binding. The immune systemmobilization structures, the second function of the antibody, areconstant domains; these regions are charged with the various effectorfunctions: stimulation of B cells to undergo proliferation anddifferentiation, activation of the complement cell lysis system,opsonization, attraction of macrophages to ingest the invader, etc.Antibodies of different isotypes have different constant domains andtherefore have different effector functions. The best studied isotypesare IgG and IgM.

[0017] The antibody itself is an oligomeric molecule, classified,according to its structure, into a class (e.g., IgG) and subclass (e.g.,IgG1). IgG molecules are the most important component of the humoralimmune response and are composed of two heavy (long) and two light(short) chains, joined by disulfide bonds into a “Y” configuration. Themolecule has two variable regions (at the arms of the “Y”). The regionsare so named because antibodies of a particular subclass, produced by aparticular individual in response to different antigens, will differ inthe variable region but not in the constant regions. The variableregions themselves are composed of both a relatively invariantframework, and of hypervariable loops, which confer on the antibody itsspecificity for a particular epitope. An antibody binds to an epitope ofan antigen as a result of molecular complementarity. The portions of theantibody which participate directly in the interaction is called“antigen binding site”, or “paratope”. The antigens bound by aparticular antibody are called its “cognate antigens”.

[0018] An antibody of one animal will be seen as a foreign antigen bythe immune system of another animal, and will therefore elicit an immuneresponse. Some of the resulting antibodies will be specific for theunique epitopes (idiotype) of the variable region of the immunizingantibody, and are therefore termed anti-idiotypic antibodies. Theseoften have immunological characteristics similar to those of an antigencognate to the immunizing antibody. Anti-isotypic antibodies, on theother hand, bind epitopes in the constant region of the immunizingantigen.

[0019] As noted above, the cells that regulate cell-mediated immunityare a class of lymphocytes called T lymphocytes. They arise ultimatelyfrom the same stem cell as B lymphocytes, however, they follow a verydifferent pathway of development in which the thymus plays an importantrole. T lymphocytes also express antigen specific surface receptorsalthough the way in which they recognize antigens is rather differentthan for B cells. T cells exist in 2 functional categories: those with aspecific effector function (cytotoxic T lymphocytes or “CTL”) and thosewith regulatory function. Regulatory T cells are required for thedevelopment of plasma cells from B cells. T helper cells (TH) produce anantigen specific up-regulation of the immune response. Immune responsescan also undergo active antigen specific down regulation. A large bodyof evidence from studies with animals and tissue culture describes thepresence of a suppressor T cell population (TS) that provides thisinhibitory regulation.

[0020] The lymphocytes in an individual specifically respond to foreignantigens but are usually unresponsive to the potentially antigenicsubstances native to that individual. Immunologic unresponsiveness isreferred to as tolerance. Self-tolerance is acquired at an earlydevelopmental stage when potentially self-recognizing lymphocytes comeinto contact with self-antigens and are prevented from developing to astage at which they would be able to respond positively to selfantigens.

[0021] The immune system has two cytokine-mediated regulatory pathwaysthat determine whether the response to antigenic challenge will beprincipally a cellular response (TH1 pathway) or principally a humoralresponse (TH2 pathway). The cellular pathway is characterized by the Thelper cell production of interleukin-2 (IL-2) or interferon-y. Thispathway mediates the delayed type hypersensitivity (DTH) response, thegeneration of cytotoxic T cells, and macrophage activation. The TH2response promotes the production by T cells of a variety of cytokines,such as interleukin-4 (IL-4) and interleukin-10 (IL-10). This responseis identified by the production of specific antibodies in high titre.

[0022] The tendency for either the cell-mediated or humoral immuneresponse to predominate is believed to be a consequence ofcross-regulation. Thus TH1 cells would inhibit the elicitation of TH2responses, e.g., by secretion of interferon-γ. Conversely, TH2-cellscould inhibit the generation of TH1-responses by producing cytokinessuch as IL-4 and IL-10.

[0023] TH2 responses might actually exacerbate the development ofcertain diseases. It is well known in the art that injections of smallamounts of immunizing antigens will preferentially elicit delayed-typehypersensitivity responses, indicative of cell-mediated immunity,whereas vaccination with larger amounts of antigen will result in a morepronounce humoral immune response as reflected by high antibody titer.However, it is difficult to avoid a high IgG response, and achieve ahigh and prolonged cellular response, by this method, and depending onthe antigen, small doses may be insufficient to elicit a sufficientlystrong CMI response to be useful.

[0024] Normally, an immune response progresses toward effectormechanisms characteristic of both B and T-lymphocytes. However, in thecourse of most immune responses, either B or T lymphocytes assume adominant role, with less substantial participation of the respectiveother type of lymphocyte. Immune responses whose effector mechanisms aremediated preponderantly through B-cells and antibodies are humoralimmune responses. Those responses wherein T-cells mediate the moreimportant effector functions are cell-mediated or cellular immuneresponses.

[0025] As noted above, the cells that regulate humoral immunity are aclass of lymphocytes called B-cells. Each clone of B-lymphocytesexpresses membrane immunoglobulins (membrane Ig's, surface-boundantibody molecules) that function as antigen receptors having one uniqueepitope for one B-lymphocyte clone. These membrane Ig molecules are thesole source of B-cell specificity. Antigens that contain an epitopecomplementary to the membrane Ig will bind to the antigen receptor. Suchantigens are also referred to as cognate antigens of the antibody.Binding to the antigen receptor (membrane Ig) will result indifferentiation and clonal proliferation of the B-lymphocyte. Some ofits progeny will differentiate into mature plasma cells which arespecialized in the synthesis of antibodies corresponding in epitopespecificity to the membrane Ig by which the B-lymphocyte had initiallybound the antigen.

[0026] The binding of an antigen to an antibody is reversible. It ismediated by the sum of many relatively weak non-covalent forces,including hydrophobic and hydrogen bonds, van der Waals forces, andionic interactions. These weak forces are effective only when theantigen molecule is close enough to allow some of its atoms to fit intocomplementary recesses on the surface of the antibody. The complementaryregions of a four-chain antibody unit are its two identicalantigen-binding sites; the corresponding region on the antigen is anantigenic determinant. Many antigenic macromolecules have many differentantigenic determinants.

[0027] For many years, live, attenuated vaccines have been used toinduce immunity against viral infections such as influenza and polio.These preparations contain live virions which cause mild, subclinicalinfections of the vaccinated individuals. In the course of suchinfections, viral vectors will enter certain host cells and code for thesynthesis of virus-specific proteins. These endogenously producedantigenic proteins are processed into smaller peptides and presented inthe context of MHC Class I and II antigens, thereby recruiting TH1 cellsand eliciting cell-medicated immune responses.

[0028] Tumor cells express certain cell surface antigens(“tumor-associated antigens”). Tumor-associated antigens are antigensthat are present in the serum and tissues of cancer patients. Many suchantigens are also expressed in embryonic tissues, and, at low levels, inthe tissue and serum of healthy individuals. Many of thetissue-associated antigens are glycoproteins, glycolipids, ormucopolysaccharides. Most tumor antigens are produced by differentiatedcells. They are produced in much larger quantities by tumor cells thanby differentiated normal cells. The human immune system recognizes thetumor antigens as native antigens and does not respond(“self-tolerance”). The mechanisms leading to self-tolerance are onlypartly understood, but it is now clear that it is largely establishedduring development of the immune system. If immature B cells or T cellsare stimulated through their antigen specific receptors at a criticalstage (e.g., just after expressing their receptors on the cell surfacebut before becoming mature), they are induced to die rather than tobecome activated. This stage occurs in the bone marrow for B cells andin the thymus for T cells. Tolerance thus will be induced toself-antigens expressed in these environments, but not to those that arenot expressed. It has been shown that normal individuals have mature Bcells capable of recognizing some self-antigens but that these B cellsare not activated. The appropriate T helper cells (TH) appear to bemissing.

[0029] For tumors that have antigens, there are at least four theorieswhy the immune response may fail to destroy a tumor: 1) there are no Bcells or cytotoxic T lymphocytes (CTL) capable of recognizing the tumor;2) there are no TH cells capable of recognizing the tumor; 3) TS cellsbecome activated before TH cells, thus preventing B-cell and CTLactivation; and 4) the genes regulating tumor proliferation may bepresent from birth, so the host does not treat the gene products as“foreign.”

[0030] Existing Solutions

[0031] Where tumor antigens appear with sufficient selectivity on atumor (i.e., the tumor antigens are absent from or present only in smallamounts on their normal cellular counterparts), the tumor antigen mayserve as a possible target for an immunotherapeutic agent.

[0032] Many of these selective tumor antigens are carbohydrate orglycoprotein (mucin) in nature. For example, most adenocarcinoma cellsabundantly express and secrete mucins. This is due in part to defects inglycosylation in cancer cells. Carcinoma cell surface mucins canphysically block immune effector mechanisms from reaching the tumor cellsurface and, therefore, the tumor antigen. That is, the host fails torecognize the tumor antigen.

[0033] In many diseases, the causative pathogens or toxins (e.g.,influenza, polio, and rabies viruses; pneumococcus bacteria; diphtheriaand tetanus toxins) can be effectively targeted and neutralized in theextracellular fluid by the mechanisms of humoral immunity throughantibodies that bind to the pathogens or toxins and thereby lead totheir inactivation of destruction. In these cases, vaccination withpreparations that elicit a humoral immune response, presumably mediatedby TH2 cells, is generally sufficient for protection. On the other hand,for many intracellular infections, for recovery from viral infections,and for targeted killing of cancer cells, it is cell-mediated immunitythat protects the organism against the invaders.

[0034] Three classes of immunotherapy are currently underinvestigation: 1) passive immunotherapy; 2) active immunotherapy withantigens; and 3) active immunotherapy with antibodies. Unfortunately,each has met with limited success. Immunotherapy, however, is preferredover antiproliferative chemotherapeutic agents, such as pyrimidine orpurine analogs, in certain stages of cancer. The analogs compete withpyrimidine and purine as building blocks used during a cell's growthcycle. The analogs are ineffective where growth is non-cycling ordormant. The majority of micrometastatic cells appear to be non-cyclingor dormant. The cytotoxic effect of immunotherapy operates independentlyof cell cycle.

[0035] “Passive immunotherapy” involves the administration of antibodiesto a patient. Antibody therapy is conventionally characterized aspassive since the patient is not the source of the antibodies. However,the term passive is misleading because the patient can produceanti-idiotypic secondary antibodies which in turn can provoke an immuneresponse which is cross-reactive with the original antigen. “Activeimmunotherapy” is the administration of an antigen, in the form of avaccine, to a patient, so as to elicit a protective immune response.Genetically modified tumor cell vaccines transfected with genesexpressing cytokines and co-stimulatory molecules have also been used toalleviate the inadequacy of the tumor specific immune response.

[0036] I. Passive Immunotherapy (with Antibodies)

[0037] A tumor antigen can serve as a reactive site to which antibodiescan become bound. Numerous antibodies have been raised against tumorantigens.

[0038] Conventional effector methods include complement dependentcytolysis (“CDC”), antibody dependent cellular cytotoxicity (“ADCC”) andphagocytosis (clearance by reticuloendothelial system after the targetcell is coated with immunoglobulin).

[0039] A relatively large quantity of antibody is required to initiateCDC, ADCC and opsonization. Furthermore, sources of human antibodies arelimited to people already suffering from the tumor of interest; it isunethical to introduce a disease into a person merely to initiateproduction of antibodies which may be harvested. As a result of thesedifficulties, antibodies of non-human origin, such as mouse antibodies,have been used.

[0040] The administration to humans of mouse antibodies, because theyare recognized as “foreign,” can provoke a human anti-mouse antibodyresponse (“HAMA”) directed against mouse-specific and mouseisotype-specific portions of the primary antibody molecule. This immunereaction occurs because of differences in the primary amino acidsequences in the constant regions of the immunoglobulins of mice andhumans. Both IgG and IgM subclasses of HAMA have been detected. The IgGresponse appears later, is longer-lived than the typical IgM response,and is more resistant to removal by plasmapheresis.

[0041] Clinically, however, HAMA: 1) increases the risk of anaphylacticor serum sickness-like reactions to subsequent administration of mouseantibodies; 2) can interfere with the immunotherapeutic effect ofsubsequently injected mouse antibodies by complexing with thoseantibodies, increasing clearance from the body, reducing tumorlocalization, enhancing uptake into the liver and spleen, and/or hidingthe tumor from therapeutic agents; and 3) can interfere withimmunodiagnostic agents and thereby hinder monitoring of the progress ofthe disease and course of treatment.

[0042] Various clinical trials have used antibodies as therapeuticagents against solid tumors. No consistent pattern of response orimproved survival has yet emerged. By contrast, antibody therapy hasmore often induced complete and long-lasting remissions in B-cell orT-cell lymphomas or leukemias. Explanations for solid tumor failuresinclude antigenic heterogeneity and insufficient accessibility ofepithelial cells to the injected antibodies as well as to secondaryeffector molecules like complement or effector cells.

[0043] As an example of passive immunity, mouse monoclonal antibody17-1A (isotype IgG2a) was used to target minimal residual disease inpatients with Duke's stage C colorectal cancer who had undergonecurative surgery and were free of manifest residual tumor. Although thetreatment improved survival and led to reduced recurrence rates, theresults were less favorable than treatment with chemotherapy alone, orin combination with radiation.

[0044] It is important to note that the target antigen for 17-1A is notshed from the membrane and is not detectable in serum. See Riethmüller,et al., “Randomized trial of monoclonal antibody for adjuvant therapy ofresected Dukes' C. colorectal carcinoma”, Lancet, 343:1177-83 (1994).

[0045] II. Active Specific Immunotherapy (“ASI”) with Tumor Antigens

[0046] ASI is defined as immunization with a defined antigen, presentedin an appropriate manner, to actively induce an immune responsespecifically to that antigen. In the context of cancer, ASI attempts tostimulate a human immune response, both humoral and cell-mediated, toattack the tumor antigen.

[0047] The humoral response and the conventional effector methods ofCDC, ADCC and phagocytosis (clearance by reticuloendothelial systemafter the target cell is coated with immunoglobulin) were discussedabove.

[0048] Over the past 5 years, considerable progress has been made in thecharacterization of the molecular complex recognized by the specificantigen receptor of T lymphocytes. Crystal structures of class I majorhistocompatibility complex (“MHC”) molecules revealed not only aputative peptide binding groove but also the actual presence in thisgroove of a peptide. After phagocytosis, proteins synthesized within thecells apparently are degraded into peptides by cellular enzymes,transported into the endoplasmic reticulum, and there, combine with theheavy chain of a class I MHC molecule. Such peptide-MHC complexes arestabilized by the addition of β2-microglobulin and transported to thecell surface where they can be recognized by the receptor of CTL. Intheory, an antigenic peptide can be derived from any intracellularprotein specifically expressed by tumor cells. See, for example, Van DerBruggen, Pierre, “The Long-Standing Quest for Tumor Rejection Antigens,”Clinical Immunology and Immunopathology, 71; 3:248-252 (1994).

[0049] III. Active Specific Immunotherapy with Antibodies

[0050] If a specific antibody from one animal is injected as animmunogen into a suitable second animal, the injected antibody willelicit an immune response (e.g., produced antibodies against theinjected antibodies—“anti-antibodies”). Some of these anti-antibodieswill be specific for the unique epitopes (idiotopes) of the variabledomain of the injected antibodies. These epitopes are known collectivelyas the idiotype of the primary antibody; the secondary (anti-)antibodies which bind to these epitopes are known as anti-idiotypicantibodies. The sum of all idiotopes present on the variable portion ofan antibody is referred to as its idiotype. Idiotypes are serologicallydefined, since injection of a primary antibody that binds an epitope ofthe antigen may induce the production of anti-idiotypic antibodies. Whenbinding between the primary antibody and an anti-idiotypic antibody isinhibited by the antigen to which the primary antibody is directed, theidiotype is binding site or epitope related. Other secondary antibodieswill be specific for the epitopes of the constant domains of theinjected antibodies and hence are known as anti-isotypic antibodies. Asused herein, anti-idiotype, anti-idiotypic antibody, epitope, orepitopic are used in their art-recognized sense.

[0051] The “network” theory states that antibodies produced initiallyduring an immune response will carry unique new epitopes to which theorganism is not tolerant, and therefore will elicit production ofsecondary antibodies (Ab2) directed against the idiotypes of the primaryantibodies (Ab1). These secondary antibodies likewise will have anidiotype which will induce production of tertiary antibodies (Ab3) andso forth.

Ab₁→Ab₂→Ab₃

[0052] The network theory also suggests that some of these secondaryantibodies (Ab2) will have a binding site that is the complement of thecomplement of the original antigen and thus will reproduce the “internalimage” of the original antigen. In other words, an anti-idiotypicantibody may be a surrogate antigen.

[0053] A traditional approach to cancer immunotherapy has been toadminister anti-tumor antibodies, i.e., antibodies which recognize anepitope on a tumor cell, to patients. However, the development of the“network” theory led investigators to suggest the direct administrationof exogenously produced anti-idiotype antibodies, that is, antibodiesraised against the idiotype of an anti-tumor antibody. Such an approachis disclosed in U.S. Pat. No. 5,053,224 (Koprowski, et al.) Koprowskiassumes that the patient's body will produce anti-antibodies that willnot only recognize these anti-idiotype antibodies, but also the originaltumor epitope.

[0054] There are four major types of anti-idiotypic antibodies. Thealpha-type binds an epitope remote from the paratope of the primaryantibody. The beta-type is one whose paratope always mimics the epitopeof the original antigen. The gamma-type binds near enough to theparatope of the primary antibody to interfere with antigen binding. Theepsilon-type recognizes an idiotypic determinant that mimics a constantdomain antigenic structure. Moreover, anti-isotypic antibodies may beheavy chain-specific or light chain-specific.

[0055] Two therapeutic applications arose from the network theory: 1)administer Ab1 which acts as an antigen inducing Ab2 production by thehost; and 2) administer Ab2 which functionally imitates the tumorantigen.

[0056] Active immunization of ovarian cancer patients with repeatedintravenous applications of the F(Ab′)₂ fragments of the monoclonalantibody OC125 was reported to induce remarkable anti-idiotypic antibody(Ab2) responses in some of the patients. Preliminary results suggestedthat patients with high Ab2 serum concentrations had better survivalrates compared to those where low or no Ab2 serum levels were detected.See Wagner, U. et al., “Clinical Course of Patients with OvarianCarcinomas After Induction of Anti-idiotypic Antibodies Against aTumor-Associated Antigen,” Tumor Diagnostic & Therapie, 11:1-4, (1990).

[0057] A human anti-idiotypic monoclonal antibody (Ab2) has been shownto induce anti-tumor cellular responses in animals and appears toprolong survival in patients with metastatic colorectal cancer. SeeDurrant, L. G. et al., “Enhanced Cell-Mediated Tumor Killing in PatientsImmunized with Human Monoclonal Anti-Idiotypic Antibody 105AD7,” CancerResearch, 54:4837-4840 (1994). The use of anti-idiotypic antibodies(Ab2) for immunotherapy of cancer is also reviewed byBhattacharya-Chatterje, et al; Cancer Immunol. Immunother. 38:75-82(1994).

DISCLOSURE OF THE INVENTION

[0058] Vaccines are preparations administered to animals or humans toeffect the prophylaxis, cure or alleviation of disease states throughinduction of specific immunity. Prophylactic vaccines are given tohealthy individuals with the intention of preparing or priming theimmune system for more effective defense against infections in thefuture. In the event of an infection or infestation, the immune systemof vaccinated individual can mount a secondary immune response and canmore rapidly recognize and eliminate the respective pathogens.Therapeutic vaccines are given to diseased individuals with the intentof stimulating or modulating the immune system which of itself haseither mounted an insufficiently effective immune response or hasaltogether failed to respond. In the design of prophylactic ortherapeutic vaccines, it is important to choose preparations that willelicit the type of immune response most capable of either providingfirst-line protection, or effecting speedy recovery.

[0059] The first step in initiating an immune response is generatinghost recognition of the tumor antigen as a foreign antigen. For example,although CA125 expression is associated with ovarian cancer, thepatient's immune system fails to recognize it as foreign. The presentinvention involves contacting a soluble antigen with a composition ofthe invention, and reacting a binding agent in the composition with thesoluble antigen. In accordance with the invention, binding the antigenwith the binding agent generates host recognition of the antigen. Inturn, generating host recognition leads to initiating an immune responseagainst the antigen.

[0060] The present invention involves the discovery that binding abinding agent to a pre-determined epitope of a multi-epitopictumor-associated antigen alters the antigen in a manner so that the hostimmune system can recognize and initiate an immune response to thepreviously unrecognized antigen. In one embodiment of the invention, abinding agent binds to a soluble tumor associated antigen, allowing thehost immune system to generate a response against the antigen. Forexample, illustrative of the present invention is B43.13, an antibodybinding agent that binds specifically to ovarian cancer antigen CA 125at the 43.13 epitope. Once B43.13 binds to the CA 125 antigen, eitherthe conformation of the antigen is altered or the antigen is processedand/or delivered differently to that it is recognized by the host'simmune system. Other examples include, but are not limited to a bindingagent that binds specifically to CA19.9, a gastrointestinal antigenassociated with gastrointestinal cancer; and to a binding agent thatbinds specifically to CA15.3, an antigen associated with breast cancer.

[0061] In accordance with the present invention, a binding agent(s) andcompositions comprising such binding agents are provided, wherein thebinding agent binds selectively to a pre-determined soluble antigen, andwherein such binding event results in the presentation of a differentepitope on the-antigen, said different epitope resulting in an immuneresponse that inhibits or kills the cells that produced the antigen.

[0062] In a preferred embodiment of the invention, a compositioncomprising a pre-determined antibody that specifically binds to apre-determined tumor associated antigen is used to bind a solubleantigen produced by the tumor. Once the soluble antigen is bound, theimmune system recognizes the antigen as “foreign,” and mounts an immuneresponse against the antigen or against the binding agent bound to theantigen. Antigens that can be made immunogenic are potentially useful toinduce or activate an immune response, leading to therapeutic andpossibly prophylactic benefits.

[0063] For diseases that can be characterized in part by having atumor-associated antigen that is multi-epitopic, the present inventioninvolves contacting a soluble antigen with a binding reagent thatspecifically binds to a single epitope on the multi-epitopictumor-associated antigen.

[0064] The binding agent may be directed against any antigen of clinicalsignificance, but preferably is directed against a tumor-associatedantigen (TAA). In the case of TAA, the cancer may include, but is notlimited to lung, colon, rectum, breast, ovary, prostate gland, head,neck, bone, immune system, or any other anatomical location. The subjectmay be a human or animal subject. Illustrative tumors and tumor markersare listed in U.S. Pat. No. 5,075,218.

[0065] The methods of the present invention involve any cancer thatproduces a soluble multi-epitopic TAA. As used herein soluble is used todescribe any antigen that is detectable in a body fluid, i.e., blood,serum, ascites, saliva, or the like. In accordance with the presentinvention, the preferred tumors are those that: shed soluble tumorantigens, e.g., tumor antigens shed into the bloodstream, as opposed toa surface antigen or an intracellular antigen; exhibit a multi-epitopictumor associated antigen, preferably of carbohydrate or glycoprotein(e.g., mucin) nature; and can be found at a concentration in thepatient's body fluid more than is normally present in healthy controlsand such a high level signifies a poor prognosis for the patient, yethas not initiated an immune response. As is well known by one skilled inthe art, one method of determining whether the concentration of the TAAis greater than is predictive of recurrence of the disease is bycomparing the patient's concentration to that of a healthy control. Ifthe concentration of the TAA is higher than the healthy control, thenthe patient's concentration is predictive of poor prognosis of thedisease.

[0066] A binding agent (BA), as used herein, refers to one member of animmunologic pair, e.g., a binding moiety that is capable of binding to asingle epitope expressed on the tumor antigen. Exemplary binding agentsinclude, but are not limited to: monoclonal antibodies (“MAb”); chimericmonoclonal antibodies (“C-MAb”); genetically engineered monoclonalantibodies (“G-MAb”); fragments of monoclonal antibodies (including butnot limited to “F(Ab)₂”, “F(Ab)” and “Dab”); single chains representingthe reactive portion of monoclonal antibodies (“SC-MAb”); tumor-bindingpeptides; any of the above joined to a molecule that mediates aneffector function; and mimics of any of the above. The antibody may be apolyclonal antibody or a monoclonal antibody. When the subject is ahuman subject, the antibody may be obtained by immunizing any animalcapable of mounting a usable immune response to the antigen, such as amouse, rat, goat sheep, rabbit or other suitable experimental animal. Inthe case of a monoclonal antibody, antibody producing cells of theimmunized animal may be fused with “immortal” or “immortalized” human oranimal cells to obtain a hybridoma which produces the antibody. Ifdesired, the genes encoding one or more of the immunoglobulin chains maybe cloned so that the antibody may be produced in different host cells,and if desired, the genes may be mutated so as to alter the sequence andhence the immunological characteristics of the antibody produced.Fragments, or fragments of binding agents, may be obtained byconventional techniques, such as by proteolytic digestion of the bindingagent using pepsin, papain, or the like; or by recombinant DNAtechniques in which DNA encoding the desired fragment is cloned andexpressed in a variety of hosts. Irradiating any of the foregoingentities, e.g., by ultraviolet light will enhance the immune response toa multi-epitopic antigen under similar conditions. In a preferredembodiment of the invention, effector functions that mediate CDC or ADCCare not required.

[0067] In an embodiment of the invention, a suitable composition for anovarian tumor associated antigen contains a binding agent that binds theCA 125 antigen. In another embodiment of the invention, a suitablecomposition for gastrointestinal cancer contains a binding agent thatbinds the CA 19.9 antigen. In yet another embodiment of the invention, asuitable composition for breast cancer contains a binding agent thatbinds the CA 15.3 antigen. Various binding agents, antibodies, antigens,and methods for preparing, isolating, and using the antibodies aredescribed in U.S. Pat. No. 4,471,057 (Koprowski) and U.S. Pat. No.5,075,218 (Jette, et al.), both incorporated herein by reference.Furtehrmore, many of these antibodies are commercially available fromCentocor, Abbott Laboratories, Commissariat a L'Energie Atomique,Hoffman-LaRoche, Inc., Sorin Biomedica, and FujiRebio.

[0068] Any composition that includes a binding agent according to theinvention may be used to initiate an in vivo immune response. Thecomposition may include one or more adjuvants, one or more carriers, oneor more excipients, one or more stabilizers, one or more imagingreagents, and/or physiologically acceptable saline. Generally, adjuvantsare substances mixed with an immunogen in order to elicit a more markedimmune response. Control vaccinations without the adjuvant resulted inhumoral immune responses. The composition may also includepharmaceutically acceptable carriers. Pharmaceutically accepted carriersinclude but are not limited to saline, sterile water, phosphate bufferedsaline, and the like. Other buffering agents, dispersing agents, andinert non-toxic substances suitable for delivery to a patient may beincluded in the compositions of the present invention. The compositionsmay be solutions suitable for administration, and are typically sterileand free of undesirable particulate matter. The compositions may besterilized by conventional sterilization techniques.

[0069] In accordance with a method of the invention, the binding agentmust contact and bind the tumor associated antigen, may be administeredto the patient by any immunologically suitable route. For example, thebinding agent may be introduced into the patient by an intravenous,subcutaneous, intraperitoneal, intradermal, intramuscular, orintralymphatic routes, in solution, tablet, or aerosol form. Liposomes,biodegradable microspheres, micelles, or the like may also be used as acarrier, vehicle, or delivery system. Furthermore, using ex vivoprocedures well known in the art, blood or serum from the patient may beremoved from the patient; optionally, it may be desirable to purify theantigen in the patient's blood; the blood or serum may then be mixedwith a composition that includes a binding agent according to theinvention; and the treated blood or serum is returned to the patient.The clinician may compare the anti-idiotypic and anti-isotypic responsesassociated with these different routes in determining the most effectiveroute of administration. The invention should not be limited to anyparticular method of introducing the binding agent into the patient.

[0070] In accordance with the present invention, the BA-antigeninteraction effectively presents the remaining epitopes to the patient'simmune system to generate: 1) a humoral response resulting in humananti-tumor antibodies that may or may not be inhibitable by the injectedantibody but are definitely inhibitable by an antibody which binds to anepitope different from the epitope reactive with the injected BA; and 2)a cell-mediated response resulting in the production of antigen-specificcytotoxic T-cells.

[0071] The binding agents of the present invention bind themulti-epitopic tumor antigen of interest, and the resulting immunogenicpair may be used to prime or initiate an immune response to anotherepitope on the antigen. As noted in more detail elsewhere in thisdisclosure, it is believed that the binding event between the bindingagent and the multi-epitopic antigen changes the conformation of theantigen sufficiently to provide access to another previouslyunrecognizable epitope on the antigen. The previously unrecognizableepitope, once recognized by agents of the immune system, initiates theimmune system cascade that results in an immune response to the wholeantigen.

[0072] In accordance with an embodiment of the invention, a cancerpatient with body fluid having endogenous, soluble multi-epitopicantigen is treated by injecting an exogenous binding agent directed to asingle epitope of the endogenous soluble antigen. After binding, theantigen reconforms or is processed and/or delivered differently allowinga different epitope on the antigen to be presented to the patient'simmune system. Upon presentation, the patient's immune system initiatesand develops a humoral, cellular, or combined humoral/cellular response,leading to tumor killing and/or stasis. Evidence of the success of thepresent invention is shown in the Examples as improved survival times.

[0073] Without intending to be bound thereby, it is believed that amechanism of action for the methods of the present invention involve aconformational alteration on the part of the soluble antigen bound by abinding agent according to the present invention. It is further believedthat binding the antigen with a binding agent directed to a firstepitope on the antigen changes the conformation of the antigensufficiently to present or activate a second epitope. It is against thissecond epitope that the patient's immune system can respond.Alternatively, the binding agent—antigen interaction may lead todifferential metabolic processing or delivery to the immune system insuch a way to activate a second epitope.

[0074] Dosage

[0075] In accordance with the methods of the present invention, acomposition comprising a binding agent may be administered in an amountsufficient to recognize and bind the pre-determined tumor associatedantigen. In a preferred embodiment of the invention, the dosage issufficient to generate or elicit an immune response against the TAA. Animmunologically or therapeutically effective or acceptable amount ofbinding agent is an amount sufficient to bind a pre-determined antigenin vivo or ex vivo, and is capable of eliciting an immune response tothe antigen. The response inhibits or kills tumor cells that carry andpresent a newly accessible epitope, thereby ameliorating or eliminatingthe disease or condition that produces the antigen. The immune responsemay take the form of a humoral response, a cell-mediated response, orboth. In a preferred embodiment of the invention, the dosage of themonoclonal antibody is less than the dosage required to elicit ADCC orCDC.

[0076] The concentration or dosage of the binding agent or active agentin the composition can vary widely, e.g., from less than about 0.01% toabout 15 to 20% by weight. As noted above, the composition isadministered in an amount sufficient to stimulate an immune responseagainst the antigen. Amounts effective for this use will depend in parton the severity of the disease and the status of the patient's immunesystem. Generally, the composition will include about 0.1 μg to about 2mg or more of binding agent per kilogram of body weight, more commonlydosages of about 1 μg to about 200 μg per kilogram of body weight. Theconcentration will usually be at least 0.5%; any amount may be selectedprimarily based on fluid volume, viscosity, antigenicity, etc., inaccordance with the particular mode of administration.

[0077] Administration may be more than once, preferably three times overa prolonged period. As the compositions of this invention may be usedfor patient's in a serious disease state, i.e., life-threatening orpotentially life-threatening, excesses of the binding agent may beadministered if desirable. Actual methods and protocols foradministering pharmaceutical compositions, including dilution techniquesfor injections of the present compositions, are well known or will beapparent to one skilled in the art. Some of these methods and protocolsare described in Remington's Pharmaceutical Science, Mack Publishing Co.(1982).

[0078] A binding agent may be administered in combination with otherbinding agents, or may be administered in combination with othertreatment protocols or agents, e.g., chemotherapeutic agents.

[0079] The effectiveness of the binding agents of the present inventionmay be monitored in vitro or in vivo. Humoral responses may be monitoredin vitro by conventional immunoassays, where the anti-tumor activity ofthe response may be determined by complement-mediated cellularcytotoxicity and/or antibody-dependent cellular cytotoxicity (ADCC)assays. The assay methodologies are well know, and are described inHandbook of Experimental Immunology, Vol. 2, Blackwell ScientificPublications, Oxford (1986). Other assays may be directed to determiningthe level of the antigen in the patient or tissue. Cell-mediatedimmunity may be monitored in vivo by the development of delayed-typehypersensitivity reactions, or other in vivo or in vitro means known tothose skilled in the art, including but not limited to the skin testreaction protocol, lymphocyte stimulation assays, measuring the toxicityof a subject's lymphocytes to tumor cells by using a standardradioactive release assay, by a limiting dilution assay, or by measuringplasma levels of IL-2 using standard ELISA assays.

EXAMPLES Example 1 Experimental Verification of the Generation ofAntibody Response Against Multiple Epitopes Present in an Antigen byInjecting an Antibody Against a Single Epitope

[0080] Cancer antigen CA125, which is expressed on more than 80% ofepithelial ovarian cancers, is used as an example to demonstrate thepresent invention.

[0081] CA125 has multiple epitopes recognized by different antibodiessuch as OC125, M11, B43.13, B27.1, among others. In the presentinvention, MAb-B43.13 was used to generate a CA125 specific immuneresponse which included recognition of the B27.1 epitope.

[0082] Method:

[0083] 86 ovarian cancer patients with active disease were tested forthe presence of antibodies against CA125. None of the patients hadantibodies against CA125 before injection of MAb-B43.13. The patientswere injected with 2 mg of MAb-B43.13 at varying time intervals (e.g.,see Table 1 for some of the patients). Sera from these patients wereanalyzed for the presence of human anti-CA125 antibodies by theirability to bind to the CA125 [R. Madiyalakan et al, Hybridoma,14:199-203 1995)]. Such anti-CA125 antibodies were further classified tobe against the B43.13 epitope or B27.1 epitope by their ability toinhibit the corresponding antibodies. The rationale for theclassification comes from the fact that anti-CA125 antibodies in thesepatients would have been generated by either of the following twopathways:

[0084] 1) If the anti-CA125 antibodies were generated in the mannersuggested by the network theory noted above, the pathway would followAb1→Ab2→Ab3. Following this scheme, MAb-B43.13 (Ab1) would generate ananti-idiotype against MAb-B43.13 (Ab2), which would in turn generate ananti-anti-idiotype against MAb-B43.13 (Ab3; or anti-CA125 antibody).Furthermore, the Ab3 antibodies generated under this pathway would bindand be inhibited only by MAb-B43.13, because the B43.13 epitope is theonly epitope present.

[0085] 2) If the anti-CA125 antibodies were generated in a mannersuggested by the present invention, the pathway would follow Ab1+solubleantigen→Ab3′. Following this scheme, MAb-B43.13 (Ab1) would bind theCA125 serum antigen, which would in turn generate an anti-CA125 antibody(Ab3′). Furthermore, the Ab3′ antibodies generated under this pathwaywould bind and be inhibited by B27.1 antibodies, because, as notedabove, CA125 is multi-epitopic and B43.13 and B27.1 epitopes aredistinct; also, Ab3′ will not bind to anti-MAb-B43.13 antibodies.

[0086] Thus, if the patients serum contained anti-CA125 antibodies thatwere inhibitable by MAb-B43.13 only, it was classified as containingAb3; those inhibitable by MAb-B27.1 were classified as Ab3′.

[0087] Results

[0088] Fourteen patients developed anti-CA125 antibodies in their sera(Table 1) in response to MAb-B43.13 injection. 10 of these 14 patientshad Ab3′ while only two patients had Ab3 antibodies in their sera. Twopatients also had both the antibodies. The presence of Ab3 in their serawas also confirmed by the ability of these antibodies to bind to thepurified rabbit anti-MAb-B43.13 antibody. There were two patients (#2and #7) who had anti-CA125 antibodies, but were not inhibitable byMAb-B43.13 or MAb B27.1, thereby suggesting that they may haveantibodies against CA125, which recognizes epitopes other than B43.13 orB27.1.

[0089] These results clearly indicate that when an antibody against asingle epitope (B43.13) was injected into a patient an antibody responseagainst the whole antigen is generated which recognizes differentepitopes present in the antigen. The presence of Ab3 in some patientscould be explained by the likely presence of excess B43.13 epitope inthe CA125 due to insufficient binding of the antibody to that epitope oridiotype induction through Pathway I. Nevertheless, the predominantmechanism of the response seems to be through Pathway II. In otherwords, injecting a monoclonal antibody to a soluble multi-epitopicantigen into a patient having a functioning immune system generates anantibody to the antigen, where the generated antibody is inhibited byantibodies to different epitopes. TABLE 1 Characterization of Anti-CA125Antibodies in Patients Injected with MAb-B43.13 Binding Inhibition [%]*Days to Anti- B27.1 Elapsed Anti- MAb- CA125 B43.13 s. F(ab')** AfterCA125 Ab B43.13 10000 chain** 1 Classi- Patient Inj.# Injection levels(Ab2)† U/mL 10 μg/mL μg/mL fication 1 3  0 14.8 + 62.3 42.6 5.8 Ab3 2 1185  9.5 − 21.6 −46.9 −86.9 Ab3' 3 3 86 25.4 + 80.2 84.4 −0.5 Ab3 3 207 48.7 + 91.4 94.0 −9.1 Ab3 4 144  79.7 + 77.1 93.0 3.5 Ab3 4 270  30.9 +79.2 83.0 −55.8 Ab3 4 309  16.7 + 77.0 83.0 −55.8 Ab3 5 134  64.1 + 89.183.3 −37.3 Ab3 4 2 15 23.6 − 62.3 −84.8 −101.9 Ab3' 2 41 21.6 − 56.920.2 −7.0 Ab3' 2 76 23.1 − 63.6 29.4 4.5 Ab3' 3 28 11.1 − 24.2 4.7 11.1Ab3' 5 1 16 15.5 + 74.8 78.3 39.9 Ab3'/A b3 6 3  0 10.3 + 54.0 60.2 22.7Ab3'/A b3 7 14.9 − 29.7 −70.2 −358.9 Ab3' 8 1  7 59.1 − 77.1 87.1 34.9Ab3' 1 17 46.9 − 78.4 86.5 40.7 Ab3' 9 3 112  9.2 − −66.4 16.0 20.2 Ab3'3 166  8.5 − −18.4 42.5 56.5 Ab3' 10  3  0 41.5 − 30.8 39.2 20.0 Ab3'11  5 134  8.8 − 19.0 24.4 3.5 Ab3' 6 134  8.7 − 18.0 39.0 46.0 Ab3' 926 13.4 − 54.5 19.3 11.1 Ab3' 9 65 13.3 − 56.1 24.4 3.7 Ab3' 10  40 9.4− 61.4 37.0 33.4 Ab3' 12  2 14 10.6 − 24.5 −54.4 19.9 Ab3' 13  1 15 11.5− 30.8 47.4 55.8 Ab3' 14  2 17 10.1 − 30.3 −51.2 1.2 Ab3'

Example 2

[0090] In pharmaceutical studies, blood samples were analyzed for CA125levels before and at selected intervals after MAb-B43.13 injection. Inpatients with elevated CA125 levels before injection, a significant dropin circulating CA125 levels could be seen immediately after MAb-B43.13injection (Table 2). This clearly demonstrated that the binding agentupon introduction into the body interacts and removes the circulatingCA125. TABLE 2 CA125 Clearance after MAb-B43.13 Injection Time (min)Patient # (CA 125 levels are givven in U/mL) after MAb 002 003 004 006007 008 010   0 760 68  65  72 90 269  431   30 210 2 7 21 16 47 141  60 144 3 0 22 16 60 79  240 240 0 0 11 15 52 97 1440 277 5 3  6 23 5996 2880 404 — 5  1 23 67 93 4320 429 — 7 — — — —

[0091] Furthermore, antigen complexed with antibody is presentedefficiently to the immune system and generates better antigen-specifichumoral and cellular response. The was demonstrated by the followingexperiments shown in Examples 3 and 4.

Example 3

[0092] Balb/c mice were immunized either with 10 μg of MAb-B43.13 inPBS, i.v.; 10,000 units of CA125 in PBS, i.v.; or 10 μg of MAb-B43.13and 10,000 units of CA125 in PBS, i.v., every three weeks for a total of3 injections. The ratio in the B43.13/CA125 injection was similar tothat observed in patients with elevated CA125 levels as determined basedon the pharmacokinetics data given in Table 2. When the mice sera wereanalyzed for anti-CA125 antibody levels, the mice Injected with theantigen-antibody complex had the highest titre. Anti-idiotype inductionin these balb/c mice are shown graphically in FIG. 1. This supports theobservation that binding agent—antigen interaction leads to betterantigen specific humoral immune response compared to binding agent orantigen alone.

Example 4

[0093] Similarly, better cellular immune response was observed when thebinding agent was presented in association with the antigen to theT-cells. Thus, macrophages isolated from mouse peritoneal cavities werestimulated with MAb-B43.13 alone; CA125 alone, a MAb-B43.13-CA125complex; or control MAb-CA125 and presented to CA125 specific mouseT-cells (isolated from mice injected with CA125). When the proliferationof T-cells as monitored by [³H]-Thymidine uptake was followed, optimalstimulation index was observed in macrophages stimulated withantibody-antigen complex (FIG. 2).

Example 5

[0094] The conclusion in Example 1 was further supported by finding acorrelation between serum CA125 levels in patients injected withMAb-B43.13 and human anti-CA125 antibody generation. The findings areshown in Table 3, and support the conclusion that the antigen should bepresent in the serum for the binding agent to interact; such interactionleads to an antigen-specific humoral response. TABLE 3 Correlationbetween Serum CA125 Levels and Antibody Levels in Patients Injected withMAb-B43.13. Anti-CA125 Antibody Titre Pre-injection Serum CA125 Level(No. of Positive/Total Patients) <100 U/mL  3/29 >100 U/mL 15/46

Example 6

[0095] The role of serum antigen in inducing multi-epitopic antibodyresponse as a consequence of an antibody injection was further confirmedin rabbit studies. Rabbits that do not contain any serum CA125, wheninjected with MAb B43.13, produced anti-CA125 antibodies that were notinhibitable by B27.1. In contrast, ovarian cancer patients with highserum antigen CA125 levels produce anti-CA125 antibodies that areinhibitable by B27.1 in response to MAb-B43.13 injection.

Example 7 Experimental Verification of Induction of Antigen SpecificAnti-Tumor Response by Antibody Injection

[0096] Human anti-CA125 antibody causes tumor cell lysis throughantibody dependent cellular cytotoxicity (“ADCC”). Although the injectedMAb-B43.13 does not cause by itself an ADCC and/or complement dependentcytolysis (“CDC”) mediated lysis of ovarian tumor cells, the generationof anti-CA125 antibodies in patients injected with MAb-B43.13, leads totumor cell lysis (see FIG. 3). This was studied in a ⁵¹Chromium releaseassay by incubating the labeled ovarian tumor cells with effector cells,and sera of six patients injected with MAb-B43.13. This supports theconclusion that the injection of a binding agent leads to itsinteraction with the antigen, with a specific humoral response resultingin anti-CA125 antibodies that cause tumor cell lysis through ADCC. Theresults clearly demonstrated the generation of antigen specificanti-tumor response after injection of the antibody.

Example 8 Generation of CA125 specific cytotoxic T-lymphocytes inpatients injected with MAb-B43.13.

[0097] Similarly injection of the binding agent to the cancer patientcontaining CA125 lead to antigen specific CTL's. Peripheral BloodMononuclear Cells (PBMC) from eight patients injected with MAb-B43.13were tested for cytotoxicity against CA125 positive or CA125 negativeovarian tumor cells in a chromium release assay. The results are shownin Table 4. The specificity of the lysis was confirmed by the ability ofMAb-B43.13 to inhibit such lysis, as well as the inability to kill CA125negative tumor cells. Of the 8 patients who received MAb-B43.13, atleast four patients (#5 to #8) were determined to have CA125 specificcytotoxic T lymphocytes (CTL's ) in their blood. The generation of CA125specific CTL's are likely to kill ovarian tumor cells in patients. TABLE4 Cytotoxicity In Patients Injected With A Vaccine Containing MAb-B43.13PERCENT DIFFERENC PERCENT BETWEEN SAMPLE INHIBITION CA 125 +ve InjectionDays Post PERCENT LYSIS BY CA 125 −ve PATIENT ID Number Injection CAOV-4SK-OV-3 K562 MAb-B43.13 (5 μg) CELLS 1 2 17  2.0 0.0 3.7 ND*insignificant 2 2 0 9.8 7.5 33.5 ND 31 3 3 0 22.8 20.4 64.3 ND 12 4 3 025.8 20.2 44.5 4.7 28 5 3 0 65.1 45.4 80.7 ND 43 6 3 0 23.1 20.0 42.019.2 16 3 6 7.4 5.2 10.2 53.0 42 7 4 355  10.3 3.1 18.9 ND 23 8 10  425 25.5 18.2 39.2 15.4 40

Example 9

[0098] Tumor killing either through an anti-CA125 antibody-mediated ADCCmechanism or through CA125-specific CLTs, lead to increased survival inpatients injected with MAb-B43.13. Although high levels of serum CA125have been suggested to be a poor prognostic indicator, they seem to havea beneficial effect in combination with the injection of anti-CA125antibody in such patients. For example, when the CA125 levels were morethan 100 units/mL, immune response against CA 125 increased by more than20% which in turn increased the median survival in those patients from39.1 months to 54.5 months (Table 5). Thus the injection of a bindingagent to a patient containing elevated levels of multiepitopic solubleantigen leads to antigen specific humoral and cellular response which inturn leads to tumor killing followed by improved survival. TABLE 5Correlation between Serum CA125 Levels, Human Anti-CA125 (Ab₁′) Responseand Survival in Patients Injected with MAb-B43.13 %-age of Patients withPreinjection Serum Human Anti- Mean Survival CA 125 Level CA 125Response in Month <100 U/mL 10.3% 39.1 >100 U/mL 32.6% 54.5

Example 10

[0099] One pancreatic cancer patient diagnosed with metastatic diseasewas repeatedly injected with a composition including an anti-CA 19.9antibody. The patient received no other treatment, and survived for 22months after the original diagnosis (19 months after surgery and theinjection) This is compared to the current survival period estimate ofsix months survival after initial diagnosis.

Example 11

[0100] Those with skill in the art recognize that the administereddosage can vary widely based on a wide set of different circumstances.The following provides preliminary dosage guidelines.

[0101] Retrospective analysis of more than 100 patients who have beeninjected up to ten times with a 2 mg dose of MAb-B43.13 indicated thatsome of these patients experienced: a) an unusual course of theirdisease, characterized by unexpectedly long survival times; and b) nosignificant adverse reaction or toxicity.

[0102] Immunological studies were conducted to understand and evaluatethe in vivo mechanism of action of MAb-B43.13. These studies indicatedthat the extent of anti-idiotypic induction in patients injected with a2 mg dose of MAb-B43.13 was unrelated to the number of injections or theclinical stage of their disease. However, anti-idiotypic induction isdependent on the levels of the circulating CA 125 present in thepatient's sera. Additional experiments demonstrated that the injectionof MAb-B43.13 into patients with measurable serum CA 125 led to theformation of antigen-antibody complexes, resulting in antigen epitopepresentation and antigen-specific humoral and cellular response to thetumor.

[0103] These studies indicate that an effective dose requires onlyenough antibody to optimally deliver and present all possiblecirculating CA 125 antigen to the immune system. In vitro studiesindicated that 1 ng of MAb-B43.13 can bind 10 units of CA 125. Assuming40 mL of plasma per kg of body weight, the injection of 2 mg ofMAb-B43.13 into a 60 kg patient can bind approximately 8333 U/mL of CA125 in serum. Since all of the ovarian cancer patients tested to datehave had far less than 8333 U/mL of CA 125 in their serum, an injectionof 2 mg of MAb-B43.13 is more than sufficient to induce the requiredimmune response. Additionally, in patients that received radiolabeledMAB-B43.13 for immunoscintographic confirmation of the disease, theresults of imaging were excellent in spite of high serum CA 125,suggesting that there is excess MAB-B43.13 for specific tumor uptake.

[0104] Furthermore, multiple injections at selected intervals appear toprovide optimal benefits to patients, since CA 125 is generatedthroughout the course of the disease.

[0105] Finally, the retrospective analysis showed that the 2 mg doseappears to have therapeutic efficacy; none of the patients (>100) havedeveloped any serious side effects or adverse reactions. If the totalHAMA response is an indication of anti-idiotypic induction, a 2 mg dosegenerates significant levels of anti-idiotypic antibodies to product thedesired therapeutic benefit. Multiple injections of 2 mg of MAb-B43.13at selected intervals appears to maintain the anti-idiotypic antibodiesat the desired therapeutic level without causing any isotypicHAMA-induced toxicity.

[0106] A range of effective doses or a therapeutically acceptable amountof MAb-B43.13 therefore includes, but is not limited to, 2 mg.

BRIEF DESCRIPTION OF THE DRAWINGS

[0107]FIG. 1 shows the superior results obtained after immunizing micewith a composition of the present invention, compared to othercompositions.

[0108]FIG. 2 shows superior macrophage stimulation caused by acomposition of the present invention, compared to other compositions.

[0109]FIG. 3 shows tumor cell lysis caused by administering acomposition of the present invention.

[0110] Industrial Applicability

[0111] The compositions comprising a binding agent according to thisinvention are particularly useful in compositions containing animmunogenic or therapeutic amount of at least one of the binding agentsof the invention. An immunogenic or therapeutic amount is an amount thatstimulates an immune response of a humoral, cellular, or combinedhumoral and cellular nature in the host. The host immune responsecomprises increased activity against an epitope on a tumor-associatedantigen that is different than the epitope to which the binding agentbinds. The compositions of the present invention are administered asanti-tumor vaccines to subjects at risk for the development of amalignancy, or to subjects showing a diagnosis of the malignancy. Thesecompositions may be used to prepare a pharmaceutical composition thatelicits an immune response.

1. A method for treating cancer comprising contacting a multi-epitopictumor-associated antigen expressed in the host serum with a compositioncomprising a binding reagent that specifically binds to a single epitopeon the tumor-associated antigen; and allowing the binding reagent tobind to the antigen to form a reagent-antigen pair, whereby theformation of the reagent-antigen pair elicits a host immune response. 2.The method of claim 1 wherein the binding reagent comprises a monoclonalantibody.
 3. The method of claim 2 wherein the target multi-epitopictumor associated antigen is CA
 125. 4. The method of claim 2 wherein thetarget multi-epitopic tumor associated antigen is CA 19.9.
 5. The methodof claim 2 wherein the target multi-epitopic tumor associated antigen isCA 15.3.
 6. The method of claim 2 wherein the tumor-associated antigenis an ovarian tumor antigen.
 7. The method of claim 1 wherein the hostimmune response is a cellular immune response.
 8. The method of claim 1wherein the host immune response is a humoral immune response.
 9. Themethod of claim 1 wherein the host immune response is both a humoralimmune response and a cellular response.
 10. A method for eliciting animmune response comprising contacting a multi-epitopic tumor-associatedantigen expressed in the host serum with a composition comprising abinding reagent that specifically binds to a single epitope on thetumor-associated antigen; and allowing the binding reagent to bind tothe antigen to form a reagent-antigen pair, whereby the formation of thereagent-antigen pair elicits a host immune response.
 11. A method forincreasing the immunogenicity of an antigen comprising contacting amulti-epitopic tumor-associated antigen expressed in the host serum witha composition comprising a binding reagent that specifically binds to asingle epitope on the tumor-associated antigen; and allowing the bindingreagent to bind to the antigen to form a reagent-antigen pair, wherebythe formation of the reagent-antigen pair elicits a host immuneresponse.
 12. A method for re-conforming a multi-epitopic tumorassociated antigen expressed in a host serum and for recognizing andinitiating an immune response, comprising contacting a multi-epitopictumor-associated antigen expressed in the host serum with a compositioncomprising a binding reagent that specifically binds to a single epitopeon the tumor-associated antigen; and allowing the binding reagent tobind to the antigen to form a reagent-antigen pair, whereby theformation of the reagent-antigen pair elicits a host immune response.13. A delivery system for a multi-epitopic tumor associated antigenexpressed in a host serum, and for recognizing and initiating an immuneresponse, comprising a composition comprising a binding reagent thatspecifically binds to a single epitope on a multi-epitopictumor-associated antigen.
 14. The method of claim 1 wherein theformation of the reagent-antigen pair elicits a host immune resposnethat overcomes tolerance of the antigen.
 15. The method of claim 1wherein the contacting step is ex vivo or in vivo.